Advanced Review The nucleolus: structure/function relationship in RNA metabolism 1 1 2 Daniele` Hernandez-Verdun, ∗ Pascal Roussel, Marc Thiry, Valentina Sirri1 and Denis L. J. Lafontaine3 The nucleolus is the ribosome factory of the cells. This is the nuclear domain where ribosomal RNAs are synthesized, processed, and assembled with ribosomal proteins. Here we describe the classical tripartite organization of the nucleolus in mammals, reflecting ribosomal gene transcription and pre-ribosomal RNA (pre-rRNA) processing efficiency: fibrillar center, dense fibrillar component, and granular component. We review the nucleolar organization across evolution from the bipartite organization in yeast to the tripartite organization in humans. We discuss the basic principles of nucleolar assembly and nucleolar structure/function relationship in RNA metabolism. The control of nucleolar assembly is presented as well as the role of pre-existing machineries and pre-rRNAs inherited from the previous cell cycle. In addition, nucleoli carry many essential extra ribosomal functions and are closely linked to cellular homeostasis and human health. The last part of this review presents recent advances in nucleolar dysfunctions in human pathology such as cancer and virus infections that modify the nucleolar organization. © 2010 John Wiley & Sons, Ltd. WIREs RNA 2010 1 415–431 INTRODUCTION nucleoli are directly related to ribosome production.4 Consequently, the size of the nucleolus is a diagnostic he nucleoli are specific nuclear domains present in marker of highly proliferative cancer cells.5 The vari- all eukaryotic cells. The nucleolus, a membrane- T ability of the nucleolar organization has been inten- less organelle, is the ribosome factory of the cell.1 In sively examined in different biological contexts such cycling cells, nucleoli assemble at the exit from mito- as proliferation, differentiation, development, and dis- sis, they are functionally active throughout interphase, ease. The comparison of the nucleolar organization and they disassemble at the beginning of mitosis. The across evolution revealed both the conservation in nucleolus is the site where different steps of ribosome the basic ‘building blocks’ and a higher complexity biogenesis are grouped together, i.e., transcription of in modern eukaryotes.6 The nucleolus constitutes a ribosomal genes (rDNAs), maturation/processing of model to understand the principles of the organization ribosomal RNAs (rRNAs), and assembly of rRNAs 2 of nuclear domains, the dynamics of protein traffick- with ribosomal proteins. It was proposed that the ing, as well as the interplay between nuclear bodies nucleolus is ‘an organelle formed by the act of building 3 dedicated to related functions (Cajal body, promyelo- aribosome’. Indeed, the organization and size of the cytic leukemia body, and nuclear speckles). Throughout the past 50 years, nucleolar com- plexity was deciphered using multiple approaches. ∗Correspondence to: [email protected] This was possible thanks to technological break- 1Nuclei and cell cycle, Institut Jacques Monod-UMR 7592 CNRS, through: specific in situ labeling, three-dimensional UniversiteParisDiderot,75205Pariscedex13,France´ resolution, and improved isolation procedures of 2Laboratoire de Biologie Tissulaire, UniversitedeLi´ ege,` Liege,` nucleoli for biochemical characterization and pro- Belgium teomic analysis. Thus, it was discovered that other 3Fonds de la Recherche cientifique (FRS-F.N.R.S.), Institut de ribonucleoproteins (RNPs) in addition to ribosomal iologie et de Medecine´ Moleculaires´ (IBMM), UniversiteLibre´ de Bruxelles (ULB), & Center for Microscopy and Molecular subunits are assembled or processed in the nucleolus. Imaging (CMMI), Academie´ Wallonie–Bruxelles, Charleroi- The best example is the nucleolar assembly proposed Gosselies, Belgium for the signal recognition particle.7 In plant cells but DOI: 10.1002/wrna.39 not in animal cells, nucleoli have been implicated as Volume 1, November/December 2010 © 2010 John Wiley & Sons, Ltd. 415 Advanced Review wires.wiley.com/rna sites of silencing RNA biogenesis.8 In addition, the comparative proteomics of animal and plant nucleoli demonstrated the nucleolar function adaptation (in humans9,10 and in plants11). Today, the nucleolus is considered a multifunc- tional domain. Extra ribosomal functions assigned to the nucleolus include the involvement in cell cycle and cell proliferation control, stress sensing and tumor surveillance pathways, apoptosis, telomere forma- tion, transfer RNA modifications, viral life-cycle, etc. Unsurprisingly, nucleolar dysfunction has severe con- sequences for human health.5,12,13 These extra ribo- somal functions of the nucleolus have been reviewed elsewhere and will not be discussed here (reviewed in Refs 14–16). This review will essentially cover three topics: (1) nucleolar organization depending on ribogenesis activity and across evolution, (2) the principles of nucleolar assembly in cycling cells, and (3) the alterations in nucleolar organization in diseases. NUCLEOLAR ORGANIZATION REFLECTS rDNA TRANSCRIPTION AND PRE-rRNA PROCESSING (a) EFFICIENCY Nucleoli assemble around the nucleolar organizer regions (NORs), as first proposed by McClintock in Zea mays.17 The NORs are chromosomal regions where multiple rDNA copies cluster in arrays. The number of NOR-bearing chromosomes varies depend- ing on the species, ranging from 1 in haploid yeast cells to 10 in human somatic cells (acrocentric chro- mosomes 13, 14, 15, 21, and 22). On mitotic chromo- somes, the active NORs are detected by a specific silver (b) staining procedure, designated Ag-NOR staining,18 FIGURE 1 Nucleolar organization of a human HeLa cell prepared reflecting their continuous association throughout by conventional| methods for electron microscopy. The cells were fixed mitosis with a subset of argyrophilic proteins belong- 19 by glutaraldehyde and osmic acid. The sections were contrasted by ing to the rDNA transcription machinery. uranyl acetate and lead citrate. (a) Section of one nucleolus and At the end of mitosis, when rDNA transcrip- (b) details of the three nucleolar components. In (a) and (b), the three tion by the RNA polymerase I (pol I) resumes (see nucleolar components are visible: the fibrillar centers (asterisks), the below), active NORs are directly involved in nucleo- dense fibrillar component (white arrow), and granular component (GC). lar reassembly. Within each active NOR, only a subset Scale bar: (a) 0.5 µmand(b) 0.1 µm. = = of rDNA units are transcribed. In contrast, inactive NORs are not bound by argyrophilic proteins, they are not associated with the pol I machinery, and they are Nucleolar Organization in Higher not involved in nucleolar formation. The nucleolus is Eukaryotes: A Tripartite Organization either organized around a single NOR or alternatively The nucleoli observed by electron microscopy (EM) several active NORs coalesce in a single nucleolus once appear to be mainly composed of fibrils and rRNA synthesis has initiated.20 For instance, in Xeno- granules on sections of fixed (formaldehyde and pus laevis or Potorous tridactilys cells, there are two osmium) samples contrasted by uranyl and lead nucleoli per cells each corresponding to a single NOR, (Figure 1(a) and (b)). A great variability of the whereas in human HeLa cells, there are two to three nucleolar morphology was described based on the nucleoli per cell corresponding to six active NORs. types or functions in animal and plant cells.2,21 416 © 2010 John Wiley & Sons, Ltd. Volume 1, November/December 2010 WIREs RNA Nucleolus FIGURE 2 Perinucleolar heterochromatin in mouse NIH3T3| nuclei observed in light microscopy and EM are shown in left and right (a) panels, respectively. (a–c) The heterochromatin is observed after DNA Dapi staining especially at the nucleolar periphery (arrows); the nucleoli (contrasted structure in phase) appeared as black holes with Dapi. (d) A protocol to preferentially reveal the nucleic acids in EM was used. The DNAs and RNAs were contrasted with (b) uranyl after methylation and acetylation of the amino and carboxyl groups. Around the nucleolus, two large clumps of chromatin (arrow heads) are visible as well as the perinucleolar chromatin. White arrows indicate intranucleolar chromatin localized in the GC. One FC is visible in the middle of the nucleolus. Scale bar 0.5 µm. EM, electron microscopy; FC, = (c) (d) fibrillar center. However, this variability resides in the arrangement FCs (number and volume) was then correlated with of three fundamental components defined by their the transcriptional activity of the rDNAs in defined texture and contrast by EM and designated fibrillar biological conditions. In the nucleolus of peripheral centers (FCs), the dense fibrillar component (DFC), mature human lymphocytes with low activity, a single and the granular component (GC) (reviewed in Ref large (diameter 0.2–0.4 µm) FC is visible.24 Upon 22). The FCs are clear fibrillar areas of different stimulation, the= lymphocytes enter the cell cycle, sizes ranging from 0.1 to 1 µmcontainingfibrils ribosome biogenesis is stimulated, and the nucleolus (Figures 1(b) and 2(d)). They are partly surrounded becomes enlarged while the numerous small FCs by the highly contrasted DFC of compact texture. are formed. It was proposed that when ribosome The FCs and DFC are embedded in the GC that production is activated, the FCs unfold because a mainly consists of granules 15–20 nm in diameter in fraction of the rDNA copies present